CLASS: 11:00 12:00Scribe: LAUREN MORRIS

CLASS: 11:00 – 12:00Scribe: LAUREN MORRIS

DATE: 9-27-2010Proof: ERIC LARSON

PROFESSOR: BARNUMCYTOKINES AND CYTOKINE RECEPTORSPage1 of 8

NO TITLE [S1]
Today we’re going to talk about cytokines
CYTOKINES [S2]
Probably every lecture we have on immunology from here on will mention a few cytokines because they are so important
The problem is cytokines are really complex
IMAGE: INFLAMMATORY RESPONSE [S3]
This is not even a current representation of what goes on and how cytokines modulate the immune response. There’s well over a hundred different cytokines and chemokines, which are a related family of molecules. It’s impossible in almost an entire semester long class to cover this stuff.
What I’m going to do today is give you a flavor of what these guys are, where they come from, how they work, a few examples of how they modulate immune responses, and then therapeutic uses because that’s becoming an increasing part in cytokines.
CYTOKINES ARE IMPORTANT BECAUSE [S4]
These guys are master regulators of the immune system,
from the cycling of stem cells to all the different progenitor cells that give rise,
to all the white blood cells, the red blood cells, megakaryocytes, platelets, on down the line,
controlling the differentiation of T cells and B cells into effector cells,
determining for B cells, which are types of antibodies they’ll make in terms of IgA, IgE, IgG, subclasses, etc…
So they really control everything in terms of getting the immune system up and running and also turning it back down to return to homeostatsis once you’ve cleared an infection.
They are becoming a very important therapeutic reagent.
Cytokine is given itself to modulate immune responses
OR an antibody is given that will sop up cytokines and prevent them from causing problems
There are lots of drugs already in use that are effective, and that list will grow and how you treat patients
CYTOKINE NOMENCLATURE [S5]
Nomenclature is a mess
When people first studied cytokines, they gave them different names based on the systems they were working in because they didn’t have an overview for how this was gonna work
You may see terms like monokines and lymphokines
originally denoted cytokines produced by only single cell types (like monocytes or activated T cells)
These terms have kind of gone by the wayside and been superceded by “interleukin”
Interleukin is a common term in almost all textbooks
Started on Interleukin 1 and now on Interleukin 37 or so
Old Definition: They were produced by leukocytes and acted on other leukocytes.
Now we know that lots of different cell types can make interleukins or can respond to interleukins because they express the appropriate receptor
One of the first types of cytokines that was identified was the interferons – mid 70’s
These are critical in controlling viral infections: virally-infected cells respond to cytokines, and these interferons tell them to do things to help eliminate the viruses
Gamma interferon is very important in firing up adaptive immune responses
There are a number of different colony-stimulating factors (CSFs) that are important in the maturation of a variety of cell types in the immune system
Granulocytes, macrophages, CSFs, monocyte-stimulating factor help B cells move from their progenitor stages to their terminally differentiated state so they can help fight infections
Chemokines have received lots of attention in the last 10-15 years
60 of them
about 5 different families
initially identified as cytokines important in the migration of leukocytes,
immune cells are constantly travelling through your body, and the blood stream moving in and out of lymph nodes and the spleen and tissues

iv. play lots of other roles too

Lots of different growth factors that turn out to be cytokines
Involved in stem cell differentiation
The terms we will hear most about are interleukins, interferons, the CSFs, and the chemokines

CYTOKINE FUNCTIONS ARE [S6]
Cytokines were difficult to figure out initially, because if people were working with B cells in culture and they found some soluble factor that mediated some function, then they named it “B Cell Factor Whatever”
If someone was doing almost the same thing with monocytes, then they called it “Monocyte something”
They began to figure out that they were actually working with the same cytokine in many cases
That showed that cytokines were pleitropic
That means they mediate lots of different functions: some of those functions can be redundant, some overlapping
We’ll discuss this in the acute phase response
Ex: Interleukin 1 can bind to a T cell and it will do something different than when an interleukin 1 binds to a monocyte or a macrophage or a neutrophil
This also depends on the context of the immune response: what’s going on, where the system is in the development of an immune response
There are lots of cytokines that are redundant in function
Ex: TNF-alpha (tumor necrosis factor) is involved in inflammation is also mimicked by interleukin 1 and interleukin 6
The interferons are redundant in function
That makes an important biological point: if you take one of these players out, then you’re in trouble; you’re gonna have a serious hole in your immune response capacity, and you’re gonna get pretty sick as a result of that
There are a few examples at the level of receptors where the receptor components are missing, and then you’re in big trouble.
Cytokines can also be synergistic
That means that when they act together, they do something more than if they were just additive
If you were looking at a response and you added interleukin 1 here (drawing on board) and you got another response when you added TNF-alpha here
if you did the same type of study and put the two together, now you’re getting something that is much greater than if you had just added the two together
They can also be antagonistic
Some cytokines help turn the immune system on, and in many cases, as soon as the immune system is turned on, the ones that are antagonistic are also produced, so they can start turning the system back down
So there are cytokines that work against each other in an effort to modulate the immune response so it doesn’t become too severe and also so it doesn’t happen at all
CYTOKINE PROPERTIES [S7]
There are some features that are fairly common for most cytokines
Most are low molecular weight proteins
about 10,000 or less MW
some a little higher
Some cytokines act only when they’re in a dimeric form, some act as trimers
These have to be in this multi-meric form in order for them to properly bind to their receptors
Most cytokines are synthesized in the active form
Some are synthesized in an inactive precursor form
Ex: An anti-inflammatory cytokine is TGF-beta: it is secreted and put out on the surface of the cell and an enzyme has to clip off a precursor part of the protein to give you the active form
Cytokines are secreted only for a brief period of time
If you look at the levels of cells that are making cytokines, the levels go up very rapidly then back down on the order of just a few hours or a few minutes
You don’t want molecules that turn out to be so potent to be continually produced
Ex: TNF-alpha is involved in toxic shock. If you made tons of TNF-alpha continually, you’d be in bad shape pretty quickly
They are active at very low concentrations
their receptors in many cases have very high affinity for the cytokines – bind very tightly, you don’t need a ton of them around in order to mediate the effects you’re looking for
Cytokines bind to very specific receptors on target cells
Cytokine actions are receptor-mediated
Without the receptor on a given cell-type, a cytokine will have no effect whatsoever
CYTOKINE RECEPTORS [S8]
Each cytokine has a receptor, but some cytokines are promiscuous and bind to more other receptors
Particularly true for chemokines, but most cytokines have a given receptor
There are at least 5 different families, not counting the chemokine family, or receptors
Many of these receptors are multi-chain complexes
In some cases there may be two or three different chains that form the receptor (Drawing on board)
Some of these receptor subunits are shared
When cytokines bind to receptors, they send signals into the cell that turns on a whole cascade of intercellular signaling events
Understand that when cytokines bind to their receptors, they turn on signaling cascades within the cell that is a whole gammit of different molecules, but they turn on certain genes and turn off certain genes as a result of their binding to their receptor
IMAGES: RECEPTOR FAMILY (A-D) [S9]
Here’s a couple of examples of some of the receptors from these 5 different families
Don’t memorize the structures! No little details,
The point: There are structurally unique families of receptors that bind to different cytokines
Immunoglobulin supergene receptors (a)
This is one that interleukin 1 will bind to
Class II receptors (I think he meant Class I image b)
Distinguished by these different domains and cysteine residues, and the conserved sequence near the plasma membrane
Binds to interleukin II
Some of the chemokine receptors are 7-membrane spanning receptors (c)
bind to G-coupled proteins intercellularly as their signals
ex: interleukin 8
Class II cytokine receptors (d)
Bind to interferons with a different receptor for each interferon
TNF receptors
Structurally have lots of different molecules, lots of different domains
TNF-alpha and beta bind to it
CYTOKINE RECEPTOR SUBFAMILIES [S10]
This is just one example of one of the subfamilies of molecules
You can see that there are two or three chains in order for the cytokine to mediate its function
Every one of these cytokine receptors has a shared subunit – the gamma chain (signaling receptor subunit)
That’s the part of the molecule that’s gonna be sending the signal down into the cell to tell the cell to do whatever it is that cytokine is responsible for
There are other portions of the molecule that actually bind to the cytokine itself (cognate receptor subunit)
You have to have all of these subunits together in order for the signal to get into the cell and the cytokine to mediate its function
Cytokine will bind to a site composed of both of these subunits
In the case of this gamma receptor, you can see it’s critical for a bunch of different cytokines
IL-2: very important in adaptive immune responses and development of T cells
IL-15
IL-7: critical for early development of lymphocytes
IL-9
IL-4
If you’re missing the gamma subunit, you can’t bind to any of these, and you’re severely immuno-compromised
GENERALIZED CYTOKINE SIGNALING MECHANISM [S11]
This is a simple diagram of how this signaling process works
This is our alpha chain for the cytokine receptor
A cognate receptor that’s gonna bind the cytokine
The beta subunit that does the signaling will also form part of that binding site
Helps to send the signal into the cell
In the Jak-stat signaling family of molecules, these kinases bind to the intercellular portion of the cytokine receptor
When they all get together like that they auto-activate, get self-phosphorylated, and they can then phosphorylate another group of intracellular proteins called the stats
When these stats get phosphorylated, they dimerize, and then they can cross the nucleus
And then these complex of these dimerized stat molecules can bind to DNA and turn on or turn off genes that are important for modulation mediated by that different cytokine
In the case of something like TNF or some other pro-inflammatory interleukins, you’re gonna turn on the expression of lots of other cytokines, you’re gonna increase the expression of adhesion molecules, turn up the expression of complement proteins…all kinds of things that these guys can do depending on the cell type that they’re interacting with
CYTOKINE EFFECTS ON TARGET CELLS ARE [S12]
How do cytokines work once they’re produced?
Autocrine interactions: the cytokine is acting on the cell that produced it
Ex: interleukin 2 for activation of T cells
When a T cell first gets an initial tweaking by binding of the T cell receptor to an antigen that’s being presented to it, that will turn the T cell on and up to start making interleukin 2
That interleukin 2 will cause an increase in the expression of its own receptor and also start activation of the T cell
Paracrine interaction: acting on nearby cells
Ex: interferons
When the cell becomes virally infected, it will start making interferon that can act on itself but can also interact with receptors on adjacent cells
If that’s the first infected cell, and its starting to make lots of virus particles, and they’re gonna be released and affect adjacent cells, if you’ve already got the danger signal out there to help control this viral infection, that’s exactly what you want
Endocrine interaction: if cytokine levels get high enough, and depending on the circumstances, they can act basically hormonal, at distant parts in the body
Ex: TNF
Large amounts of TNF made in a really serious bacterial infection, blood levels can go up quite a bit, (this is bad)
When you have fever, when you have an infection, that means cytokines have been dumped into the blood stream, reached the part of the brain that controls temperature, and modulated that
HEMATOPOIETIC CYTOKINES IN THE IMMUNE SYSTEM [S13]
A few examples of how cytokines work at different stages of the immune system; how they handle various types of infections
Cytokines are very important in stem cell generations
So stem cell factor is a cytokine involved in keeping stem cells recycling
A pool of stem cells is always available so you can make more cells
When you get lots of infection or you have a serious infection, the pool of cells that handle those gets depleted
Some of the pro-inflammatory cytokines that are generated later in infection can come back and help modulate the effects
Once they begin to become progenitors, there are a variety of cytokines that push these progenitors to their terminally differentiated cell type
There’s a common lymphoid progenitor
There’s controversy in how this works
One of the key cytokines in the development of T cells and B cells is Interleukin-7
Without IL-7, these will never mature and exit the thymus or the bone marrow
Lots of other cytokines are important in their subsequent development
There are lots of cytokines that will push progenitors to be any number of differentiated cells like neutrophils, monocytes, mast cells, basophils…
Lots of times we have some idea of what is critical for that
To make neutrophils and monocytes you need colony-stimulating factors
IL-3 is very important in helping that process where you split down different lines
CYTOKINES ARE CRITICAL FOR LYMPHOCYTE DEVELOPMENT [S14]
Recapitulation of the idea of IL-7 being very critical in the development of B and T cells
B cells are maturing in the bone marrow (in stromal cells)
They get signals as they’re developing from adhesion molecules, stem cell factors, etc.
Once they begin to express the receptor for IL-7, that’s a critical step in their maturation process
It’s the very stem cells that they’re binding to that are producing that IL-7 so that they can go on to become Pre-B cells and eventually immature B cells and begin expressing immunoglobulin on their surface
If they don’t get this signal, they’re not gonna mature to the next stage
T CELL MATURATION AND CYTOKINES [S15]
T cells also need lots of cytokines. IL-2 is very critical for activation, for proliferation, and growth of those cells after antigen binding
T cells also get clonally selected once their T cell receptor encounters antigen, they become activated, they’re gonna proliferate lots of those T cells around, just like you’ll have lots of specific B cells around in response to an antigen
Depending on the environment (the type of antigen and the other cytokines and factors that are gonna be generated during an immune response) these T cell subsets can turn into many different flavors
Th1 and Th2 are some of the more commonly known ones
Th1 type cytokines (gamma-interferon and IL-2) – these are involved in cell-mediated immunity
Th2 type cytokines (IL-4, IL-5, and others) – involved in antibody-mediated immune responses
A lot of attention on Th17 T cells because they produce IL-17 molecule that seems to be very important in innate immune responses for helping to activate neutrophils
B CELL MATURATION AND CYTOKINES [S16]
B cells also need lots of different cytokines
IL-6, one of the pro-inflammatory cytokines, is very important for their growth – won’t mature without it
Certain cytokines are very critical for the development of certain types of immune responses
Transforming-growth factor-Beta (TGF-is important in telling the B cells to start making IgA (IgA antibodies in oral cavity/gut/lungs – don’t want a lot of inflammation in these areas
TGF- is considered an anti-inflammatory cytokine, but also augments an antibody that might bind to a bacteria or virus that’s causing problems in those tissues
IL-4 is very important for IgE which is important in the development of allergies
Let’s walk through what cytokines do in various types of infections…
CYTOKINES IN HOST DEFENSE VIRAL INFECTIONS [S17]
Viruses are pathogens that bind to a receptor on some cell, and they’re internalized
they need the host cell machinery to reproduce themselves and then other cells
when they get inside they start replicating their RNA or DNA right away, depending on the type of virus they are
It’s the viral genetic material that is recognized by the innate immune response, the toll-like receptors
Ex: TLR3 or TLR8 are good at recognizing viral genetic material (dsRNA or ssRNA) that then tweaks the cell to start making interferons
These are the anti-viral cytokines that are critical to clearing up viral infections
VIRAL RESPONSIVE TLRS [S18]
What do they actually do? These are the TLRs
Structurally similar – leucine repeats
Differ in binding sites at the terminal portion
INTERFERONS [S19]
There are two types of Interferons – Types I and II
Type I is alpha and beta combined
Produced by all cells in the body because all cells can be infected with a virus
So they all have the capacity to produce a cytokine to help eliminate infections in that cell and surrounding cells
Type II is gamma interferon
More limited in its production – largely by activated T cells
Produced later down the line in the viral infection
These are important in clearing the viruses and in activating the T cells, particularly the cytotoxic T cells that come in a lyse the virally infected cells
Question: Did you say what a TLR is?
Toll-like receptor
There are whole families of toll-like receptors
Critical in kicking off the innate immune response
Recognize the foreign structures on bacteria and viruses, parasites, and pathogens
INTERFERON ANTI-VIRAL MECHANISMS [S20]
Interferons eliminate viruses first by inducing the expression of these MHC molecules
MHC molecules bind small peptides derived from invading pathogens and express them on the surface of the antigen presenting cells so it can interact with the appropriate T cell to get the adaptive immune response kicked off
In the case of viral infections, the MHC molecule we’re talking about is MHC-Class I
This takes in its binding site a small peptide derived from that virus, puts it up on the surface of the cell, and then a cytotoxic T cell that’s specific for that peptide will then recognize that virally infected cell and kill it
MHC expression increased provides more peptides on surface and better chance of T cell recognizing that virally infected cell and eliminating it
A number of viruses have figured this out, so they quickly shut down the expression of MHC, so T cells can’t recognize the infected cell
We have a second backup system: Natural Killer (NK) Cells
They wander around the body all the time and survey all the cells in the body
If a cell is not expressing MHC-Class I molecules, it recognizes something is wrong and kills it
Interferons also shut down the cell that has been infected
Induces the production of RNases that degrade viral mRNA, but it can also degrade cell mRNA too
Produce proteins that block the function of molecules involved in protein synthesis
Virus can’t make its proteins, but cell can’t either

iii. So it becomes a death signal for the cell, killing the cell and virus with it